Many current diagnostic in-vitro assays are sold in kit form to the final user, who must use the purchased kit within a specified shelf life of the product. Many assay kits are used to assay serum or plasma for a particular component. Many kits have controls that contain human or bovine sera and reagents that are used during the assay to react with test serum or plasma.
Since kits are dated with a specific shelf life, the various components of the kit must be protected against microbial contamination for the shelf life through the use of preservatives or antimicrobials. Unfortunately, some well known preservatives cannot be used in certain diagnostic assay kits. For example, sodium azide cannot be used with horseradish peroxidase conjugates because it interferes with the heme group of the horseradish peroxidase. Gentamicin is ineffective against fungi. Thimerosal contains mercury which cannot be used in certain countries, such as Japan, because of its undesirable health and environmental effects.
Esters of p-hydroxybenzoic acid are known to be effective against both bacteria and fungi, but data suggests that in diagnostic assays, their efficacy may be blocked by serum proteins. These esters also have low solubility in aqueous systems, making formulating with them difficult. In particular, alkyl p-hydroxybenzoate esters, also known as parabens, have demonstrated antimicrobial properties. At low concentrations, parabens exhibit broad spectrum antimicrobial activity against gram positive and qram negative bacteria, and against fungi. Currently the parabens are used primarily in the food and cosmetic industries. See Gottfried, N.S., Alkyl p-Hydroxybenzoate Esters as Pharmaceutical Preservatives (A Review of the Parabens), Am. J. Hosp. Pharm. 19:310-314 (1962).
Substituted 4-oxo-l,4-dihydroquinoline-3-carboxylic acids and substituted 4-oxo-l,4-dihydro-l,8-naphthyridine-3-carboxylic acids, known as quinolones, are antibacterials that are used clinically as antibiotics, and are not known to be fungicidal or fungistatic. Aryl-fluoroquinolones, particularly ciprofloxacin, ofloxacin, enoxacin, norfloxacin, difloxacin and the quinolone commonly known as A-56620 have been evaluated for both in-vivo and in-vitro antibacterial activity. A-56620 is structurally similar to norfloxacin, except for a p-fluorophenyl substitution at position 1. All of the above aryl-fluoroquinolones exhibit various levels of antibacterial activity to aerobic bacteria. See, Fernandes, P. B. et al., In Vivo Evaluation of A-56619 (Difloxacin) and A-56620: New Aryl-Fluoroquinolones, Antimicrobial Agents and Chemotherapy 29:201 (1986); Barry, A. L. et al., In Vitro Evaluation of A-56619 and A-56620, Two New Quinolones, Antimicrobial Agents and Chemotherapy 29:40 (1986). However, when A-56620 and difloxacin were evaluated for antibacterial activity using a culture broth containing 50% serum, the potency of these quinolones decreased by an average of 4-fold for A-56620 and from 4- to 64-fold for difloxacin. Stamm, J.M. et al., In Vitro Evaluation of A-56619 (Difloxacin) and A-56620: New Aryl-Fluoroquinolones, Antimicrobial Aqents and Chemotherapy, 29:193 (1986).
Although both the aryl-fluoroquinolones and the parabens exhibit broad spectrum antimicrobial activity, the aryl-fluoroquinolones against bacteria and the parabens against bacteria and fungi, both show a loss of potency with serum based test systems.
An antimicrobial that is stable over the shelf life of the in-vitro diagnostic assay kit, that is effective in the presence of serum or plasma or other proteinaceous material, and which has broad spectrum antimicrobial activity against fungi and bacteria would minimize the problems currently associated with in-vitro diagnostic assay kits. A need also exists for a method of protecting these assay kits against microbial contamination.